Intraosseous delivery of lentiviral vectors for hemophilia A gene therapy

用于血友病 A 基因治疗的慢病毒载体骨内递送

• 批准号: 9329473
• 负责人: Carol H Miao
• 金额: $ 69.77万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9329473
• 关键词: Address; Alpha Granule; Animal Model; Animals; Antibody Formation; Apoptosis; Automobile Driving; Biological; Blood Circulation; Blood Platelets; Bone Marrow; Bone Marrow Cells; Bypass; CD34 gene; Canis familiaris; Cells; Cessation of life; Clinical; Complication; Development; Disease susceptibility; Dose; Elongation Factor; Engraftment; Ensure; F8 gene; Factor VIII; Factor VIIa; Gene Expression; Gene Transfer; Glycoproteins; Hematopoietic stem cells; Hemophilia A; Hemorrhage; Hemostatic Agents; Hemostatic function; Human; Immune Tolerance; Immune response; In Vitro; Infusion procedures; Injury; Innate Immune Response; Lentivirus Vector; Megakaryocytes; Modeling; Morbidity - disease rate; Mus; Patients; Pattern; Phenotype; Plasma; Platelet Activation; Platelet Count measurement; Property; Protocols documentation; Quality of life; Regimen; Resistance; Risk; Role; Safety; Site; Stem cells; Tail; Testing; Therapeutic; Therapeutic Effect; Thrombocytopenia; Variant; Viral; antibody inhibitor; cost; cytokine; effective therapy; gene therapy; humanized mouse; improved; in vitro Assay; in vivo; inhibitor/antagonist; integration site; mouse model; new technology; novel; novel strategies; pre-clinical; preconditioning; prevent; promoter; response; targeted treatment; vector

PROJECT SUMMARY Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, ~25% of treated patients develop anti-FVIII immune responses (inhibitors); it is particularly challenging to treat these inhibitor patients. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-FVIII antibody formation is highly desired for patients with or without inhibitors. Recently it was shown that FVIII expressed ectopically in megakaryocyte (Meg) is effective to prevent HemA mouse tail bleeding to death. Ex vivo HSC gene therapy corrected the bleeding diathesis even in the presence of inhibitors. This is because FVIII synthesized in Megs is stored in α-granules and only released at the injury sites during platelet activation, therefore protected from circulating inhibitors. However, several limitations exist for ex vivo gene therapy. First, FVIII stored in platelets (pFVIII) has different temporal-spatial availability compared with plasma FVIII. pFVIII was shown to have variable efficacy in different hemostasis mouse models and its resistance to inhibitors also varies in different settings. The functional roles of gene therapy delivered pFVIII and its resistance to inhibitors will need to be carefully investigated and defined. Second, there are difficulties encountered by ex vivo HSC gene therapy, in particular, preconditioning required by ex vivo gene therapy is highly undesirable for HemA patients. Third, it was shown that high levels of pFVIII expression can induce platelet apoptosis. Preconditioning regimens used by ex vivo gene therapy induce thrombocytopenia. This problem can be compounded with pFVIII expression, leading to significant thrombocytopenia, which poses a major hemostatic risk to HemA inhibitor patients. Recently, intraosseous (IO) delivery of lentiviral vector (LV) has been shown to effectively transduce bone marrow (BM) cells in mice. We propose to employ this new approach to deliver F8- LVs into BM, which avoids the use of preconditioning regimen, thereby decreasing the associated risk with thrombocytopenia and can be both safe and efficacious for clinical use. Previously, we demonstrated a single IO delivery of F8-LVs driven by a human Meg-specific glycoprotein 1bα (GP1bα) promoter (G-F8-LV) produced platelet-specific FVIII expression, leading to long-term, partial correction of HemA mice both with and without pre-existing inhibitors. In the current proposal, we will expand our studies targeting FVIII expression in platelets via IO delivery, aiming at optimizing this novel technology and developing preclinical protocols in humanized mice and large animal models (HemA dogs). AIM 1. Optimize IO delivery of G-F8-LVs in mice and examine the resulting biological efficacy of pFVIII. AIM 2. Optimize pFVIII gene expression in human Megs. AIM 3. Evaluate if IO delivery of LVs can effectively transduce HSCs in large animal models and if IO delivery of canine F8 (cF8)-LV can correct the phenotype in HemA dogs.

项目概要 目前对甲型血友病 (HemA) 患者的治疗方法是反复输注因子 VIII (FVIII；缩写为 F8（构建体中的 F8）成本昂贵、不方便且不完全有效。此外，约 25% 的接受治疗的患者 产生抗 FVIII 免疫反应（抑制剂）；治疗这些抑制剂患者尤其具有挑战性。 基因治疗可实现长期表型校正，且不会出现抗 FVIII 抗体并发症 对于有或没有抑制剂的患者来说，形成是非常需要的。最近显示FVIII表达 异位注入巨核细胞（Meg）可有效防止HemA小鼠尾部出血致死。离体HSC 即使存在抑制剂，基因治疗也可以纠正出血素质。这是因为FVIII Megs 中合成的储存在 α 颗粒中，仅在血小板激活期间在损伤部位释放， 因此免受循环抑制剂的影响。然而，离体基因治疗存在一些局限性。第一的， 与血浆 FVIII 相比，储存在血小板中的 FVIII (pFVIII) 具有不同的时空可用性。 pFVIII 在不同的止血小鼠模型中显示出不同的功效，并且其对抑制剂的抵抗力也不同 在不同的设置中有所不同。 pFVIII 基因治疗的功能作用及其对抑制剂的耐药性 需要仔细研究和定义。二、离体HSC遇到的困难 基因治疗，特别是离体基因治疗所需的预处理对于 HemA 来说是非常不可取的 患者。第三，显示高水平的pFVIII表达可以诱导血小板凋亡。 离体基因治疗使用的预处理方案会引起血小板减少症。这个问题可以是 与 pFVIII 表达复合，导致显着的血小板减少，这是一个主要的止血剂 HemA 抑制剂患者的风险。最近，慢病毒载体（LV）的骨内（IO）递送已被证明可以 有效转导小鼠骨髓 (BM) 细胞。我们建议采用这种新方法来交付 F8- LV 进入 BM，避免使用预处理方案，从而降低相关风险 血小板减少症，临床使用安全有效。之前我们演示了一个 由人 Meg 特异性糖蛋白 1bα (GP1bα) 启动子 (G-F8-LV) 驱动的 F8-LV 的 IO 递送 血小板特异性 FVIII 表达，导致 HemA 小鼠的长期、部分校正，无论是否有 预先存在的抑制剂。在当前的提案中，我们将扩大针对血小板中 FVIII 表达的研究 通过IO交付，旨在优化这项新技术并开发人性化的临床前协议 小鼠和大型动物模型（HemA 狗）。目的 1. 优化 G-F8-LV 在小鼠体内的 IO 递送并检查 pFVIII 的生物学功效。目标 2. 优化人类 Megs 中的 pFVIII 基因表达。目标 3. 评估 如果 IO 递送 LV 可以在大型动物模型中有效转导 HSC，如果 IO 递送犬 F8 (cF8)-LV可以纠正HemA狗的表型。